Antibacterial Activities of a New Brominated Diterpene from Borneon Laurencia spp

In our continuous interest to study the diversity of halogenated metabolites of Malaysian species of the red algal genus Laurencia, we examined the chemical composition of five populations of unrecorded Laurencia sp. A new brominated diterpene, 10-acetoxyangasiol (1), and four other known metabolites, aplysidiol (2), cupalaurenol (3), 1-methyl-2,3,5-tribromoindole (4), and chamigrane epoxide (5), were isolated and identified. Isolated metabolites exhibited potent antibacterial activities against clinical bacteria, Staphylococcus aureus, Staphylococcus sp., Streptococcus pyogenes, Salmonella sp. and Vibrio cholerae.


Introduction
Red algae of the genus Laurencia (Rhodomelaceae, Ceramiales) are known to be prolific sources of a wide variety of halogenated secondary metabolites, such as C 15 -acetogenins and C 15 -, C 20 -, and C 30 -terpenoids [1]. In the course of our chemical and biological investigation of Laurencia species from the coastal waters of Borneo (Malaysia), we reported the chemical composition of L. snackeyi (Weber-van Bosse) Masuda [2], L. similis Nam et Saito [3], and L. majuscula (Harvey) Lucas [4,5]. As part of the chemical analysis of the undescribed Laurencia species, we examined five populations of OPEN ACCESS unrecorded Laurencia sp. collected from the coastal waters of North Borneo Island, Sabah. Each specimen contained one halogenated metabolite, a total of five halogenated metabolites were isolated and identified. These specimens yielded one new 10-acetoxyangasiol (1) and four known halogenated metabolites; aplysiadiol (2), cupalaurenol (3), 1-methyl-2,3,5-tribromoindole (4), and chamigrane epoxide (5). The structure of the new compound 10-acetoxyangasiol (1) was elucidated by spectral data. The structures of the known metabolites (2)(3)(4)(5) were determined based on the comparison of spectral data to that of the published reports of Ojika et al. [6,7], Ichiba and Higa [8] and Carter and Rinehart [9]. In this paper, we describe the isolation and structure elucidation of these compounds and their antibacterial activities against clinical bacteria.

Results and Discussion
The partially dried specimens of algae Laurencia sp. were extracted in MeOH (1:1, v/v). The concentrated extracts were partitioned between H 2 O and EtOAc. The EtOAc soluble fraction was dehydrated over Na 2 SO 4 anhydrous, filtered, concentrated and purified by a combination of silica gel column chromatography and High Performance Liquid Chromatography (HPLC) separation, to yield compounds 1-5 ( Figure 1).
Compound 1 was obtained as white powder, [] 25 D +6.4 (c 0.9, CHCl 3 ). The IR spectrum indicated the presence of OH group (3532 cm -1 ), -lactone carbonyl group (1773 cm -1 ) and acetoxy functionality (1734 and 1236 cm -1 ). The positive ESI-MS exhibited a characteristic molecular-ion cluster at m/z 535/537/539 in a ratio of 1:2:1, suggesting the presence of two bromine atoms. The molecular formula was determined to be C 22 H 32 Br 2 O 5 by HR-ESI-TOFMS, indicating six degrees of unsaturation. The 13 C NMR spectrum ( According to the molecular formula and the functionalities mentioned above, compound 1 is suggested to contain one -lactone and three carbocyclic rings. Furthermore, the 13 C-NMR spectra of 1 closely resembled those of the known compound angasiol [10], except for the absence of one acetoxy group. It clearly suggested that 1 possesses the same skeleton and substituent.   Figure 2 were consistent with the proposed structure of 1. Furthermore, the relative stereochemistry of 1 was elucidated by NOESY experiments as well as the coupling constants in the 1 H-NMR spectrum. The coupling constant (J 6,10 = 8.9 Hz) indicated the anti conformation for the H-6/H-10. The configuration at C-10 was assigned as R* on the basis of the NOESY correlations observed between H-5/H-10, H-7/H-10, H-10/Hax-12, H-10/Hax-16 and H 3 -17/H 3 -Ac. In addition, the remaining stereochemistry of 1 was determined by NOESY correlations as shown in Figure 3 and assigned to be the same as for angasiol and its related compounds, irieols A-G [10][11][12]. In consequence, the structure of 10-acetoxyangasiol (1) must be represented by structure 1.
All five metabolites were subjected to antibacterial bioassay against five species of clinical bacteria, and their antibacterial activities at 30 mg disc -1 are shown in Table 2. Compounds 1, 2 and 3 exhibited potent inhibition against three of the tested bacteria. The lowest MIC value was observed for compound 1 against Vibrio cholerae at 100 g mL -1 . Compounds 4 and 5 only exhibited weak inhibition against Staphylococcus sp. with a MIC value of 300 g mL -1 . Vancomycin, used as a positive control, exhibited a >23 mm inhibition zone against all the tested microbes at 30 mg disc -1 . Based on our findings, these halogenated metabolites could be considered as possible candidates for further investigation against clinical microbes in our endeavor to combat the rise in antibiotic resistant microbes.

General
Optical rotations were measured on an AUTOPOL IV automatic polarimeter (Rudolph Research Analytical). 1 H-NMR (600 MHz) and 13 C-NMR (150 MHz) spectra were recorded with a JEOL ECA 600, with TMS as internal standard. HR-ESI-TOFMS spectrum was obtained with LCMS-IT-TOF (Shimadzu). Silica gel (Merck, Kieselgel 60, 70-230 mesh) was used for column chromatography. Separation of the eluted fraction were carried out using Shimadzu HPLC with Phenyl Hexyl (Phenomenex, USA) 10 x 250 mm eluted with 70% MeCN/H 2 O, and detected at 210 nm using SPD 20A Shimadzu UV-Vis detector. Analytical TLC was performed on Merck Kieselgel 60 F 254 . Spots were visualized by UV light or by spraying with a 5% phosphomolybdic acid-ethanol solution.

Bioassay
The antibacterial bioassay for the isolated metabolite was carried out against 5 species of clinical pathogens obtained from Department of Pathology, Queen Elizabeth Hospital, Kota Kinabalu, Sabah, Malaysia. These bacteria are Staphylococcus aureus (UMS01-08), Staphylococcus sp. (UMS02-08), Streptococcus pyogenes(UMS03-08), Salmonella sp. (UMS04-08) and Vibrio cholerae(UMS05-08). One loopful of each organism was precultured in 20 mL of peptone water overnight. The turbidity of the culture was adjusted to an optical density (OD) of McFarland 0.5 [13]. Then 0.1 mL of the precultured bacterial suspension was used to seed Nutrient Agar plates. Paper discs (Whatman, 6 mm) impregnated with 30 mg disc -1 of the respective isolated compounds were placed on the seeded agar plates and the diameter of the inhibitory zones measured after incubation at 28 C for 24 h. Antibacterial activity was evaluated by measuring the diameter of inhibition zone of the tested bacteria. Vancomycin (Sigma, Germany) was used as positive control. Minimum Inhibitory Concentration (MIC) determination for the positive inhibitions was carried out via microdilution broth method as described by Shan et al. (2008) with slight modifications [14].

Conclusions
This is our first report on the composition of halogenated metabolites and their activities in unrecorded Laurencia sp. from the coastal waters of North Borneo Island of Sabah, Malaysia. Our initiative pertaining to the isolation and identification of halogenated secondary metabolites from Borneon Laurencia continues to excite us, with the isolation of a wide diversity of structurally interesting metabolites. To date, we have isolated a total of 42 halogenated metabolites from L. snackeyii, L. majuscula, L. similis, L. nangii and these five species of undescribed Laurencia spp.